Abstract

Understanding the genetic and molecular bases of gene function is of increasing importance to harness their potential to produce plants with novel traits. One important objective is the improvement of plant productivity to meet future demands in food crop production. Gene function is mostly characterized through overexpression or silencing in transgenic plants. This approach is a lengthy procedure, especially in cereals. Plant viral expression systems can be used for rapid expression of proteins. However, current systems have a small cargo capacity and have mostly been used for gene silencing. Here, a four-component barley stripe mosaic virus-based system with high cargo capacity was constructed for the rapid and stable expression of recombinant proteins in different plant species, allowing function analyses at different stages of development. Fluorescent marker proteins are expressed at high levels within 1 week, and a proof of efficient function analysis is shown using the aluminum malate transporter1 gene. In addition to the ability of gene cotransformation, this work demonstrates that the four-component barley stripe mosaic virus-based system allows the overexpression of cDNAs of up to 2,100 nucleotides (encoding a protein of ∼78 kD), thereby providing an invaluable tool to accelerate functional genomics and proteomic research in monocot and dicot species.

Gene function characterization is essential to identify genes and gene variants that play important roles in plant development or adaptation/responses to environmental cues in a changing and unstable climate. To meet future demands in food crop production, agricultural production will have to increase by at least 60% by 2050 to feed a world population projected to rise to over 9 billion people (Godfray et al., 2010; Tilman et al., 2011; Ray et al., 2013). Transgenic plants represent an important tool for gene studies by gene overexpression or silencing, since they bring information for decision making in breeding programs (Job, 2002; Varshney et al., 2005). Genetic transformation by Agrobacterium tumefaciens and biolistic-based systems are still the two main approaches used to integrate exogenous DNA into the genomes of several crops (Vasil et al., 1992; Cheng et al., 1997; Barampuram and Zhang, 2011). However, these methods result in variable efficiencies of plant transformation depending on plant species, with random integration of new sequences into the plant genome resulting in varying levels of gene expression in different lines (Taylor and Fauquet, 2002; Bhalla et al., 2006; Kim et al., 2007). Moreover, transgenic plant production cannot yet be considered to be a routine practice, since this generally requires the ability to combine efficient transformation and regeneration of whole plants from transformed tissues (Halpin, 2005). Furthermore, the generation of transgenic plants requires genotyping and/or backcrossing of several regenerated plants in order to obtain the desired mutants. These steps are labor and cost intensive and cannot be afforded by all laboratories. This time-consuming step also is a bottleneck hard to overcome, and this is especially the case for monocotyledonous crop species (Barampuram and Zhang, 2011). The first CRISPR-Cas9-engineered wheat (Triticum aestivum) plants were obtained at the Chinese Academy of Sciences (Zhang et al., 2016). It has been reported that from 1,600 bombarded immature embryos, only 80 mutants were identified, suggesting that a considerable amount of time and labor is required in functional characterization of one or several gene candidates. Although novel methods based on the CRISPR-Cas9 technology are being developed to produce plants with novel traits (Cao et al., 2016), this technology still requires plant regeneration from genome-edited transformed material. Genome complexity and time-consuming tissue culture could be a challenge discouraging scientist to undergo ambitious genome-editing projects without proof of function of the gene to be edited. A rapid system to evaluate gene function would greatly speed up gene validation and selection prior to their use in transgenic crops or breeding protocols.

Plant viruses are powerful tools in which to express heterologous proteins. They can be used as vehicles to drive high levels of gene expression throughout the plant within a short time frame (Hefferon, 2014). Many plant viruses, including Tobacco mosaic virus, Potato virus X, and Tobacco rattle virus, have successfully been designed and developed as vectors to express foreign proteins in dicotyledonous plants (Chapman et al., 1992; Baulcombe et al., 1995; Jia et al., 2003; Lu et al., 2003; Hefferon, 2014). Despite these advances, there is currently no suitable virus vector for systemic expression of heterologous proteins in the recalcitrant monocotyledonous plants. In recent years, the barley stripe mosaic virus hordeivirus (BSMV) was adapted for virus-induced gene silencing to study gene function in monocots (Purkayastha and Dasgupta, 2009; Senthil-Kumar and Mysore, 2011; Yuan et al., 2011). BSMV is known to infect major crop plants. The BSMV genome is composed of a tripartite positive-sense RNA virus (RNAα, RNAβ, and RNAγ; Jackson et al., 1989). RNAα and RNAγ are essential for the viral genome replication, whereas RNAβ encodes the coat protein and movement proteins. RNAγ is bicistronic and encodes an RNA polymerase component (γa protein) and a Cys-rich γb protein involved in viral pathogenicity (Lee et al., 2012). The understanding of its main protein functions has led to the development and improvement of different BSMV systems for ease of use and high-throughput expression (Jackson et al., 2009; Pacak et al., 2010; Yuan et al., 2011).

A few studies have reported successful use of the BSMV system for virus-mediated overexpression (VOX) of a small protein such as a fungal toxin (ToxA, 534-bp cDNA, encoding a protein of 19.7 kD; Tai et al., 2007; Lee et al., 2012). Furthermore, we recently reported that inoculation via an imbibition method allows a rapid, uniform, and efficient overexpression of a small fluorescent protein named iLOV (improved light, oxygen, or voltage sensing; Chapman et al., 2008) in wheat (336-bp cDNA; Cheuk and Houde, 2017). This approach showed promising potential for the investigation and characterization of small proteins. However, current BSMV systems have a constraint in terms of gene size, since studies involving the GFP (720-bp cDNA) as reporter revealed patchy GFP expression throughout infected tissues (Haupt et al., 2001; Lawrence and Jackson, 2001). Other studies reported that the insertion of fragments in the 150- to 500-bp range (maximal protein size of ∼18 kD) into the BSMV γ genome is relatively stable and maintains higher chances for systemic infection (Holzberg et al., 2002; Bruun-Rasmussen et al., 2007; Scofield and Nelson, 2009). This suggests that larger fragments may limit the systemic spread of BSMV due to instability in the viral genome (Bruun-Rasmussen et al., 2007; Lee et al., 2012). Geminivirus vectors known to infect a wide range of plant species, including wheat, have been developed in recent years for heterologous protein expression. As for other viral systems, the cargo capacity of the inserted gene is very limited in the original viral system. However, the insertion of larger foreign genes is possible and involves the suppression of important genes coding for movement protein and coat protein, thereby eliminating cell-to-cell movement or plant-to-plant transmission and repressing viral replication (Zaidi and Mansoor, 2017). Previously, a BSMV modification comprising a four-component BSMV vector has been suggested to allow the expression of larger proteins (Lee et al., 2012). However, there has been no follow-up on this suggestion. Given the limitation of existing viral vectors to express foreign genes, we demonstrate in this study the ability of a four-component BSMV system to overexpress genes of larger size within 7 d post infection (dpi). Using the aluminum malate transporter gene (TaALMT1; 1,380 bp) known to improve Al tolerance in transgenic plants (or an ALMT-GFP fusion protein of 2,106 bp), we show that the viral system allows gene function characterization, giving results that are similar to previous studies using transgenic plants (Delhaize et al., 2004; Pereira et al., 2010). As an additional feature, the availability of a four-RNA system allows the expression of two different proteins in planta. Another attractive aspect of BSMV is its ability to infect many different plant species (Jackson and Lane, 1981), allowing the rapid evaluation of gene effects in various species and cultivars without the tedious production of transgenic plants or gene subcloning into different vectors.

RESULTS

Modification of BSMV Vectors for Gene Overexpression

To demonstrate the potential of the four-component BSMV system for the expression of large proteins in planta, we first modified the γ genome to produce the γ1 and γ2 genomes and included ligation-independent cloning (LIC) sites with an ApaI restriction enzyme site for easier cDNA cloning. The new four-component BSMV system composed of vectors encoding the α, β, γ1-, and γ2-modified genomes (Fig. 1; for details, see Supplemental Fig. S1) was then used for gene overexpression and compared with the standard three-component BSMV system (Yuan et al., 2011).

Schematic representation of the four-component BSMV system. In the pCaBS-α, pCaBS-β, pCaBS-γ1, and pCaBS- γ2 vectors, the α, β, γ1, and γ2 cDNAs were cloned between the double cauliflower mosaic virus 35S promoter and a ribozyme sequence (Rz) in the pCass4-Rz plasmid. A LIC cloning site containing an ApaI site was inserted into the γ1 and γ2 genome to substitute the γb and γa genes from the original γ genome, respectively. The lethal ccdB gene was inserted into the ApaI restriction enzyme site of the LIC site to facilitate subcloning and the selection of recombinant plasmid. For details of vector construction, see Supplemental Figure S1.

As a first step, we verified that the γa and γb genes, expressed respectively by the γ1 and γ2 genomes, are able to function in trans-configuration for virus replication and gene overexpression. The coding region of the iLOV fluorescent marker was inserted into the γ1 or γ2 LIC site, then wheat seeds were infected with the four-component system (α, β, γ1:iLOV, and γ2 or α, β, γ1, and γ2:iLOV) by our seed imbibition method (Cheuk and Houde, 2017). Strong iLOV fluorescence was observed within 5 d in infected roots whether the cDNA was inserted in γ1 or γ2 (Fig. 2, A and B), demonstrating successful infection and propagation of the virus with appropriate gene expression. If we omit one of the two modified γ RNAs, the expression of iLOV is reduced greatly, indicating that the four components (α, β, γ1, and γ2) are required for efficient heterologous protein expression and viral propagation (Fig. 2, C and D). The need to maintain both γ1 and γ2 RNAs opens the possibility of expressing two different genes at the same time.

Heterologous expression of the iLOV fluorescent reporter protein in wheat using the four-component BSMV system. Schematic representations of the constructs used for inoculation by seed imbibition are shown, and fluorescence was detected by confocal microscopy at 5 dpi (A–D) or 7 dpi (E). A and B, Expression of γ1-expressed or γ2-expressed iLOV in root, respectively. C and D, Expression of iLOV in the absence of γ2 or γ1 in root, respectively. E, Coexpression of mCherry and GFP in root and leaf. Closeup images are shown to observe individual cells (at right). Rz, Ribozyme sequence.

The Four-Component BSMV Allows the Coexpression of Two Genes

Coexpression, often achieved via particle bombardment or agroinfiltration of immature embryos in wheat, is an important technique for the study of protein interactions (Yao et al., 2007; Wang et al., 2015, 2017). However, these approaches are time consuming when the production of transgenic plants expressing two genes is desired. An efficient tool allowing the fast study of protein interactions in planta remains a major challenge, especially in cereals. To verify that the four-component BSMV system can express two different proteins simultaneously, cDNAs of the mCherry and GFP fluorescent reporters were inserted into RNAγ1 (γ1:mCherry) and RNAγ2 (γ2:GFP), respectively. Wheat seeds were inoculated with the four components α, β, γ1:mCherry, and γ2:GFP (Fig. 2E) by imbibition. Confocal fluorescence microscopy showed strong mCherry (magenta) and GFP (green) fluorescence of infected wheat leaves and roots at 7 dpi. The merged images (Fig. 2E, white) reveal colocalization of expression and little or no variation in the expression/stability of the two proteins (mCherry and GFP) in different cells. This is observed in the merged images by the presence of green or magenta in a few cells, while most of the merged image is white, indicating similar expression of both proteins. This indicates that the four-component BSMV vector system is able to successfully coexpress two genes in planta.

The Three-Component BSMV System Has a Low Cargo Capacity

Transformation using the three-component BSMV system and the leaf abrasion inoculation method to express GFP was shown to yield patchy fluorescence throughout the infected leaf tissue (Lawrence and Jackson, 2001; Lee et al., 2012), suggesting the instability of large inserts within the γ genomic BSMV RNA (Scofield and Nelson, 2009; Lee et al., 2012). Recently, we demonstrated that a seed imbibition method leads to a more uniform and rapid expression of the smaller iLOV marker compared with the leaf abrasion inoculation method (Cheuk and Houde, 2017). The uneven GFP signal distribution reported in previous studies (Lawrence and Jackson, 2001; Lee et al., 2012) could be due to the inoculation method and/or instability caused by the insert size within the RNAγ genome. To verify the cargo limit of the three-component BSMV system, the iLOV (336 bp) and GFP (720 bp) cDNAs were inserted into the γ genome vector or in the γ2 genome vector (Fig. 3). The three-component BSMV system, comprising pCaBSα, pCaBSβ, and pCaBSγ:iLOV or pCaBSγ:GFP (Fig. 3A), and the four-component BSMV system, comprising pCaBSα, pCaBSβ, pCaBSγ1, and pCaBSγ2:iLOV or pCaBSγ2:GFP (Fig. 3B), were then used to inoculate wheat by seed imbibition. At 7 dpi, the iLOV or GFP fluorescence was detected in root or leaf tissues by confocal microscopy. While the iLOV fluorescence signal is comparable between the three- and four-component BSMV systems (Fig. 3C), the GFP signal is much lower in root and leaf tissues (Fig. 3D). The closeup images show little or no signal in many cells using the three-component BSMV system to express GFP. These results confirm and extend previous reports regarding the cargo limit of the original three-component BSMV VOX system (Holzberg et al., 2002; Bruun-Rasmussen et al., 2007; Scofield and Nelson, 2009).

The cargo capacity of the three-component BSMV system is lower than that of the four-component BSMV system. A and B, Schematic representations of the three-component (BSMV 3 RNA) and four-component (BSMV 4 RNA) systems, respectively. C and D, Detection of iLOV and GFP expression by confocal microscopy in wheat roots and leaves at 7 dpi following inoculation with two systems by seed imbibition. Closeup images are shown to observe individual cells (at bottom). Rz, Ribozyme sequence.

The Four-Component BSMV System Allows Overexpression of TaALMT1 in Sensitive and Tolerant Wheat Cultivars

Overexpression of the aluminum-activated malate transporter TaALMT1 in wheat confers aluminum tolerance by protecting roots from Al toxicity (Delhaize et al., 2004; Sasaki et al., 2004). To verify that the four-component BSMV system is suitable for the expression of a functional protein in planta, TaALMT1 was cloned into γ1 (γ1:TaALMT1) and γ2 (γ2:TaALMT1) vectors. The four-component BSMV system was then used to inoculate Al-sensitive (cv NIL 107 and NIL 108) and Al-tolerant (cv Atlas 66) wheat cultivars by seed imbibition. At 5 dpi, total RNA was extracted from roots and the copy number of TaALMT1 transcripts was quantified by qRT-PCR (Table I). In all experiments, we used uninfected wheat and wheat infected with empty vector (BSMV:00) as controls, and the results were similar between these two controls (Table I). Our results revealed that the TaALMT1 transcript was poorly expressed in Al-sensitive cultivars (cv NIL 107 and NIL 108, less than 200 copies of TaALMT1 transcripts per 10 ng of total RNA) compared with the Al-tolerant cv Atlas 66 (around 5,000 copies) in uninfected or BSMV:00-infected plants (Table I). Following inoculation with the four-component system comprising γ1:TaALMT1, the number of TaALMT1 transcripts increased significantly to 2,994 and 2,250 copies in cv NIL 107 and NIL 108, respectively, and to 10,249 copies in cv Atlas 66. When wheat seeds were inoculated with γ2:TaALMT1, a higher abundance of TaALMT1 transcripts was detected. The copy number of TaALMT1 transcripts was 5,804 and 5,911 in cv NIL 107 and NIL 108, respectively, and 19,898 in cv Atlas 66. Cotransformation (γ1:TaALMT1 and γ2:TaALMT1) increased the transcript copy number to 9,222 and 9,340 in cv NIL 107 and NIL 108, respectively, and to 26,536 in cv Atlas 66. To evaluate whether the TaALMT1 transcripts are functional and to determine whether the amount of transcript is correlated with Al tolerance, we examined the effects of TaALMT1 overexpression on root growth under Al toxicity. At 5 dpi, inoculated seedlings of cv NIL 107, NIL 108, and Atlas 66 were exposed to different Al concentrations (0, 5, 25, 50, 100, 250, or 500 μm) for 24 h. At 5 μm Al, the root growth inhibition (RGI) in uninfected or BSMV:00-infected cv NIL 107 and NIL 108 seedlings was 42% and 49.6%, respectively. Inoculation with γ1:TaALMT1 resulted in a decrease of RGI (Table I) compared with the controls. In cv NIL 107 and NIL 108 seedlings, the RGI decreased to 24.7% and 24.2%, respectively, when exposed to 5 μm Al, indicating an increase in tolerance. When seedlings were inoculated with γ2:TaALMT1 or coinoculated with γ1:TaALMT1 and γ2:TaALMT1, the RGI decreased further to 2.5% and 5.3% at 5 μm Al in cv NIL 107 and NIL 108, respectively. This improvement of Al tolerance also is observed at higher Al concentrations. When the cv NIL plants were cotransformed with γ1:TaALMT1 and γ2:TaALMT1, they were as tolerant as cv Atlas 66, with a 50% RGI of around 50 µm. Roots continued to grow even at 100 µm Al. These results confirm that TaALMT1 overexpression can help the sensitive cultivars to achieve the Al tolerance of their parent cv Atlas 66. However, a slightly higher copy number of TaALMT1 is required in cv NIL 107 and NIL 108 to reach a similar Al tolerance (Table I). This suggests that other genes that contribute to Al tolerance are not expressed appropriately in the cv NIL lines compared with their parent.

TaALMT1 transcript copy number in root tips of control plants (not exposed to Al) was estimated by qRT-PCR and normalized against 18S rRNA at 5 dpi. The transcript number was calculated using a standard curve with known amounts of TaALMT1 cDNA. The different constructs shown indicate in which γ vector the TaALMT1 gene was inserted. The other γ vector complement was empty when not mentioned. The root growth inhibition was measured after 24 h of Al exposure. Data show means ± sd (n = 90 roots). For total RNA extraction, each biological replicate contained approximately 30 root tips. Dashes indicate that this condition was not evaluated.

In the Al-tolerant cv Atlas 66, a 50% RGI was obtained with 50 μm Al in uninfected or BSMV:00-infected plants, and strong inhibition was observed after exposure to 100 μm Al. In contrast, plants inoculated with γ1:TaALMT1, γ2:TaALMT1, or cotransformed with γ1:TaALMT1 and γ2:TaALMT1 were tolerant to 250 μm Al, with RGIs of 61.9%, 50.2%, and 35.2%, respectively. At an even higher Al concentration (500 μm), the RGI of plants inoculated with γ1:TaALMT1 and γ2:TaALMT1 was still only 58.2%, demonstrating that the known Al-tolerant cv Atlas 66 can become much more Al tolerant by increasing the level of TaALMT1 expression.

The Four-Component BSMV System Has a High Cargo Capacity

The large deletion of the γa coding sequence (1,947 bp) in γ2 was predicted to allow stable expression of genes of about 2 kb. To verify this hypothesis, a TaALMT1-GFP construct (2,106 bp) was inserted into the γ2 genome (Fig. 4A). γ2:TaALMT1-GFP was inoculated in wheat seeds, and the presence of TaALMT1-GFP in seedlings at 7 dpi was verified by reverse transcription (RT)-PCR (Fig. 4B). GFP tagging of TaALMT1 enables visualization of the progression of the four-component BSMV VOX with the large insert (2,106 bp) in infected plants using confocal microscopy. Strong GFP fluorescence was detected throughout the root and the leaf at 7 dpi in both Al-tolerant (cv Atlas 66) and Al-sensitive (cv NIL 107 and NIL 108) cultivars (Fig. 4C). The presence of the TaALMT1-GFP fusion protein in infected roots and leaves (predicted at 78 kD) was confirmed by immunoblotting using an anti-GFP antibody (Fig. 4D). Plants overexpressing GFP were used as a control to confirm the fusion (Fig. 4D, γ2:GFP). Furthermore, quantitative RT-PCR showed that TaALMT1-GFP is expressed strongly (Fig. 4E), as observed for TaALMT1 (Table I). The functionality of TaALMT1 as a GFP fusion protein was tested in Al-sensitive (cv NIL 107 and NIL 108) and Al-tolerant (cv Atlas 66) cultivars by exposing inoculated plants to Al for 24 h. A 50% RGI was reached at 25 and 250 µm Al in the Al-sensitive and Al-tolerant cultivars, respectively (Fig. 4E), showing that the GFP tag does not affect the activity of the TaALMT1 transporter (Table I, γ2:TaALMT1; RGI at 25 µm Al in cv NIL 107 and NIL 108 is 53% and 52.4%, respectively, while in cv Atlas 66 exposed to 250 µm Al, the RGI is 50.2%). The GFP fluorescence distribution was then monitored at 14 and 30 dpi to verify the stability of TaALMT1-GFP expression. As shown in Figure 4F, the GFP signal remained stable at 30 dpi in leaf and root tissues. Since BSMV is able to infect the reproductive organs and can be transmitted through seeds (Jackson et al., 2009; Cheuk and Houde, 2017), we also verified that the four-component BSMV system can express the reporter gene in different tissues (Fig. 5, A–H) as well as in the progeny of infected plants (Fig. 5, I and J).

Expression of TaALMT1-GFP in wheat using the four-component BSMV system. A, Schematic representations of the constructs used for seed imbibition. Rz, Ribozyme sequence. B, RT-PCR analysis of TaALMT1-GFP, BSMV γ1, and BSMV γ2 transcript accumulation in uninfected plants and plants infected with BSMV:00 (α, β, empty γ1, and empty γ2) or the γ2:TaALMT1-GFP-harboring BSMV system (with empty γ1 as described in A) at 7 dpi. C, Detection of TaALMT1-GFP expression by confocal microscopy in roots and leaves of different wheat cultivars at 7 dpi. D, Immunoblot analysis of GFP expression in leaf and root tissues inoculated with four-component BSMV systems harboring empty γ1 and γ2 vectors (BSMV:00), a γ2:TaALMT1-GFP vector, or a γ2:GFP vector at 7 dpi. E, Quantification of TaALMT1 transcript copy number in roots and leaves of uninoculated plants and plants inoculated with BSMV:00 or γ2:TaALMT1-GFP at 5 dpi (left side of each graph). Determination of root growth inhibition followed 24 h of Al exposure (i.e. at 6 dpi). Different letters indicate statistically significant differences between samples (P < 0.05 by Tukey’s test). F, Detection of GFP fluorescence from the TaALMT1-GFP construct in leaves and roots by confocal microscopy in leaves and roots at 30 dpi.

Expression of TaALMT1-GFP in different tissues/organs of γ2:TaALMT1-GFP infected wheat plants. A to H, Closeups show individual cells in root (A), radicle (B), flag leaf (C), closeup on flag leaf tip (D), crown (E), anther (F), pollen (G), and seed (H). I and J, A seed from the first generation of plants (T0) was grown to observe the expression of TaALMT1-GFP in the first leaf (I) and a root (J) of the next generation of γ2:TaALMT1-GFP-infected wheat plants at the seedling stage (7 d).

Longer cDNAs, TaALMT1-GFP-iLOV (2,448 bp) and β-galactosidase (2,799 bp), also were tested in the four-component system (cloning in the γ2 genome). However, only weak GFP fluorescence and no difference in β-galactosidase activity were detected in infected plants (data not shown), suggesting an instability of larger inserts. Considering that the TaALMT1-GFP (2,106 bp) cDNA is properly expressed, we conclude that the size limit of the cDNA to be expressed and properly propagated by the four-component BSMV system is between 2,106 and 2,448 bp.

The Four-Component BSMV System Can Be Used in Different Monocot and Dicot Species

Since BSMV is able to infect many monocot and dicot plant species (Jackson and Lane, 1981; Holzberg et al., 2002; Cheuk and Houde, 2017), we investigated the ability of the four-component system to inoculate two different monocot and dicot species. Secale cereale, Brachypodium distachyon, Arabidopsis thaliana, and Nicotiana benthamiana were inoculated with the α, β, γ1, and γ2:TaALMT1-GFP vectors by seed imbibition. At 7 dpi, the presence of the TaALMT1-GFP insert was verified in roots and leaves using RT-PCR (Fig. 6A). The high GFP fluorescence indicated uniform expression of TaALMT1-GFP in the four species (Fig. 6B). Moreover, we performed similar tests to verify the application of the system in a broader range of plant species. We show that the four-component BSMV system can infect all 12 additional plant species obtained from a local store (Supplemental Fig. S3).

Expression of TaALMT1-GFP using the four-component BSMV system in different monocotyledonous and dicotyledonous species. A, Detection of transcripts in uninfected, BSMV:00-infected, or γ2:TaALMT1-GFP-infected S. cereale, B. distachyon, N. benthamiana, and A.thaliana at 7 dpi. B, Detection of GFP fluorescence from the TaALMT1-GFP construct by confocal microscopy at 7 dpi.

DISCUSSION

Transformation methods are remarkably difficult and time consuming in cereals (Hiei et al., 2014). So far, no efficient vectors have been engineered to allow the overexpression of large proteins in planta for functional studies. The three-component BSMV system has been shown to stably overexpress small genes such as ToxA (534 bp; Tai et al., 2007) and iLOV (336 bp; Cheuk and Houde, 2017). Another interesting system based on the Wheat streak mosaic virus has been reported to efficiently and stably express GFP in wheat (Tatineni et al., 2011). However, it has not yet been proven whether this system could allow the expression of larger proteins in wheat or other cereals. Based on the efficient machinery and the known genome structure of BSMV, we generated a new BSMV system composed of four RNA components that improved cargo capacity (Fig. 1). Using this new viral system, we were able to rapidly express functional heterologous proteins within a short time frame (5–7 d). The efficiency of the system was verified by inserting a small fluorescent protein (iLOV) in either the γ1 or γ2 genome (Fig. 2). While the four-component system gave strong fluorescence of iLOV throughout the infected root tissues (Fig. 2, A and B), the deletion of either γ1 or γ2 led to a weak green fluorescence (Fig. 2, C and D). This demonstrates the importance of the four RNA components for efficient heterologous protein expression in this system. Furthermore, the successful expression of GFP using the four-component BSMV system suggested that this system can exceed the limit of the original system in terms of cDNA size (Fig. 3D). We demonstrated that the four-component BSMV system allows the expression of larger cDNAs with a size limit between 2,106 bp (TaALMT1-GFP fusion protein, properly expressed) and 2,448 bp (TaALMT1-GFP-iLOV, weakly expressed).

The function of the overexpressed protein was verified using this system. Transgenic studies using Al-sensitive wheat plants overexpressing TaALMT1 under the control of the constitutive ubiquitin promoter demonstrated that this gene can confer an Al tolerance level similar to that of an Al-tolerant wheat (Pereira et al., 2010). Several tests using the TaALMT1 gene were performed as a proof of concept. The overexpression of TaALMT1 conferred Al tolerance to Al-sensitive cultivars, giving comparable results to studies involving transgenic wheat plants overexpressing TaALMT1 (Pereira et al., 2010). So far, there have been no studies evaluating the effect of TaALMT1 overexpression in a tolerant wheat cultivar. This is likely due to the labor-intensive process required to perform this experiment. In addition, overexpression under the control of a strong ubiquitin promoter may have been considered as the maximal level of expression possible, indicating that there was a low probability of further improving Al tolerance with this gene in a tolerant cultivar. Our expression system allowed us to evaluate the impact of TaALMT1 overexpression in the Al-tolerant wheat cv Atlas 66. We demonstrated that the overexpression of TaALMT1 in this cultivar greatly enhanced tolerance to Al with another 10-fold increase in Al tolerance (Table I). This indicates that the ease of use of this system allows rapid and efficient transformation to evaluate gene effects in various genetic backgrounds or cultivars.

Furthermore, the four-component BSMV system allows simultaneous coexpression of two genes when the cDNAs are cloned in the γ1 and/or the γ2 genome (Fig. 2E). This system could thus be an efficient tool for the evaluation of gene dosage effects in planta. The use of this system also could facilitate protein interaction studies using fluorescent proteins with different spectral properties and provide a novel system in which to perform Förster resonance energy transfer analysis in various plant species. The possibility of adding protein tags (at the N or C terminus) in this expression system may facilitate studies that aim to identify protein-interacting partners in planta by coimmunoprecipitation after infection. On the other hand, the four-component BSMV system opens up the possibility of studying genes that are expressed in various tissues and plant species during early development (as early as 5 dpi) or at later stages during flowering or seed maturation. The reduced time to study protein function facilitates the study of gene variants or mutated versions to understand or improve protein function. This new BSMV system combines different advantages compared with other transformation systems: ease of use, high transformation efficiency, broad plant host range (17 species tested in this study), reduction in cost, ability to express larger genes in different tissues, and maintenance of overexpression in the next generation. These results suggest that the four-component BSMV system is an attractive tool for gene expression in a broad range of plant species, as shown for the three-component BSMV system (Jackson and Lane, 1981). This technology provides an invaluable and powerful tool for prescreening and the selection of candidate genes aiming to accelerate plant functional genomics and proteomics research.

MATERIALS AND METHODS

Construction of a Four-Component BSMV System with LIC Cloning Sites

The vectors forming the three-component BSMV system were kindly provided by Dawei Li. The pCaBS-α, pCaBS-β, and pCaBS-γ vectors bear the cDNAs encoding the α, β, and γ genomic RNAs of the BSMV strain ND18 (Yuan et al., 2011). The pCass4-Rz Agrobacterium tumefaciens-compatible Ti plasmid (Annamalai and Rao, 2005) was selected for the construction of the pCaBS-γ1 and pCaBS-γ2 vectors in this study (Supplemental Fig. S1, A and B).

The γ1 and γ2 cDNAs were PCR amplified with the BS-24/BS-26 primer pairs with the Q5 high-fidelity DNA polymerase (New England Biolabs), giving blunt-end PCR products. The γ1 and γ2 cDNAs were then digested with BamHI and inserted between StuI and BamHI sites of the pCass4-Rz binary vector under the control of a double 35S promoter. This generated the pCaBS-γ1 and pCaBS-γ2 vectors, respectively. To facilitate the subsequent subcloning of genes of interest, a ccdB lethal gene was inserted in the ApaI sites of the LIC-γ1 and LIC-γ2 elements to produce pCaBS-γ1:ccdB and pCaBS-γ2:ccdB, respectively (Fig. 1; Supplemental Fig. S1, C and D). These pCaBS-γ1:ccdB and pCaBS-γ2:ccdB plasmids were propagated in Escherichia coli DB3.1.

The LIC strategy was used for efficient cloning of the coding sequences of iLOV (330 bp), GFP (720 bp), mCherry (711 bp), TaALMT1 (1,380 bp), TaALMT1-GFP (2,106 bp), TaALMT1-GFP-iLOV (2,448 bp), and β-galactosidase (β-gal; 2,799 bp). The iLOV coding sequence was PCR amplified from pGEX iLOV (Addgene; plasmid no. 26587) using the iLOV-F1/iLOV-γ1 or iLOV-F1/iLOV-γ2 primer pair. The TaALMT1 (TaALMT1-1; accession no. AB081803) cDNA was amplified by RT-PCR from total RNA isolated from roots of Triticum aestivum (cv Atlas 66) using the TaALMT-1_PCR-F1/TaALMT-1_PCR-R1 primer pair followed by amplification of the first PCR products using the nested primers TaALMT-1_PCR-F2 and TaALMT-1_PCR-R2. This amplified PCR product was cloned in the γ1 or γ2 vector using the TaALMT1-F1/TaALMT1-γ1 or TaALMT1-F1/TaALMT1-γ2 primer pair, respectively. TaALMT-1 was then sequenced in both vectors to confirm that the sequence did not contain mutations. The mCherry coding sequence was amplified from pFA6a-link-yoPA-mCherry-Kan (Addgene; plasmid no. 44950) using the mCherry-F1/mCherry-γ1 primer pair. The GFP coding sequence was amplified from PAVA319 plasmid (ABRC stock no. CD3-374) using the GFP-F1/GFP-γ2 primer pair. To generate the TaALMT1-GFP fusion protein, TaALMT1 and GFP (without start codon) were fused by overlap extension PCR in which a four-Gly peptide (Gly-Gly-Gly-Gly) was inserted as a linker between the two genes. The same strategy was used to generate the TaALMT1-GFP-iLOV fusion protein. The β-gal coding sequence was amplified from p155 pCMV-EGFP/β-gal (Addgene; plasmid no. 8387) using the βgal-F1/βgal-γ2 primer pair. Details of the primers used to amplify iLOV, GFP, mCherry, TaALMT1, TaALMT1-GFP, and β-gal are described in Supplemental Table S1.

LIC Procedure

The pCaBS-γ1:ccdB and pCaBS-γ2:ccdB LIC-containing vectors were digested with ApaI, which removed the ccdB gene (Supplemental Fig. S1, C and D). Genes of interest were PCR amplified, purified, and cloned into pCaBS-γ1 or pCaBS-γ2 by LIC as described (Supplemental Fig. S1, C and D). The T4 DNA polymerase-treated vector (200 ng of pCaBS-γ1 or pCaBS-γ2) was mixed with each of the T4 polymerase-treated inserts at different vector-insert molar ratios, incubated at 66°C for 2 min, and slowly cooled at room temperature. The annealed products were then transformed into competent DH10B E. coli cells. Colonies were screened by colony PCR and restriction mapping, and proper inserts were confirmed by sequencing.

Agroinfiltration of N. benthamiana and Viral Inoculation

Viral particles to be used for imbibition are produced in N. benthamiana by agroinfiltration. The pCaBS-α, pCaBS-β, pCaBS-γ or pCaBS-γ1, and pCaBS-γ2 were transformed individually into A. tumefaciens strain EHA105, as described previously (Yuan et al., 2011). Equal amounts of the three or four A. tumefaciens strains (OD600 = 0.7) were mixed and incubated for 3 to 5 h at 28°C. Agroinfiltration of N. benthamiana leaves was performed using a 1-mL needleless syringe. After infiltration, plants were maintained in a controlled-environment chamber. At 7 dpi, 0.5 g of spot-agroinfiltrated leaf was harvested and ground in 1 mL of 20 mm sodium phosphate buffer (pH 7.2) using a mortar and pestle. The resulting homogenate contained approximately 2.85 × 1011 copies of BSMV per 1 mL of homogenate that can be used directly for viral inoculation or divided into aliquots in small volumes and stored at −20°C for later use. Seeds of different species were used for seed imbibition. If commercial seeds are used, they may be coated with fungicides. If so, the seeds can be washed on an orbital shaker for 6 h in distilled water with water changes at every 30 min before viral inoculation. The viral particles were diluted 1:100 with distilled water before viral inoculation. For large seeds, the imbibition was performed for 3 d in a petri dish by covering the seeds to half of their height. For some very large seeds that did not germinate in petri dishes (H. annuus and P. sativum), imbibition was initiated in moist vermiculite and the seeds were transferred to a petri dish with viral particles as soon as they started to germinate. They were then transferred in vermiculite for short-term growth or in a mixture of soil:perlite:peat moss (2:1:1) for long-term growth. For small seeds, the imbibition was performed on 1% agarose gels (in water) in petri dishes. Seeds were deposited on agar plates, and the viral particles (100 µL for a 60-mm petri dish) were inoculated over the seeds.

Aluminum Exposure and Root Growth Measurement

After 2 d of imbibition in BSMV-loaded N. benthamiana homogenate, wheat seeds were transferred to moist vermiculite and allowed to germinate for an additional 3 d. Seedlings were carefully washed with deionized water to remove vermiculite. Trays were filled with 3 L of solution containing 1 mm CaCl2 (pH 4.15) and different Al concentrations ranging from 0 to 500 μm as described for each experiment. Seedlings (10 per tray) were exposed to Al at 5 dpi for 24 h. Root elongation was measured with a ruler at 0 and 24 h. The RGI is expressed as 100 × [1 − (root growth of Al-treated seedlings divided by the root growth of control seedlings)].

Quantitative RT-PCR Analysis

Root or leaf tissues were frozen directly on dry ice for RNA isolation. Total RNA, which includes viral RNA, was isolated using the RNeasy Plant Mini Kit (Qiagen) and treated with on-column RNase-free DNase. After assessing quality on agarose gels, RNA samples were reverse transcribed to cDNAs with iScript Reverse Transcription Supermix (Bio-Rad) according to the manufacturer’s instructions. Real-time quantitative RT-PCR was performed on a CFX96 Touch Thermal cycler (Bio-Rad) using the SsoFast EvaGreen Supermix (Bio-Rad). The data were exported as Excel files for analysis. The copy number of iLOV and TaALMT1 was normalized to the 18S rRNA and determined from a calibration curve using known amounts of iLOV and TaALMT1 cDNAs, respectively. iLOV, TaALMT1, and GFP regions were amplified using the respective primer pairs: iLOV-F2/iLOV-R2, GFP-F2/GFP-R2, and TaALMT1-F2/TaALMT1-R2 (for primer sequences, see Supplemental Table S1). The quantitative PCR run program consisted of a first step at 95°C for 5 min followed by 40 cycles of 15 s at 95°C and 30 s at 58°C.

Detection of iLOV, GFP, TaALMT1, and TaALMT1-GFP by RT-PCR

Total RNA was extracted from approximately 7-d-old seedlings and reverse transcribed to cDNA as described above. The following primer pairs were used to detect iLOV, GFP, TaALMT1, TaALMT1-GFP, γ1 genome, and γ2 genome, respectively: iLOV-F2/iLOV-R2, GFP-F2/GFP-R2, TaALMT1-F2/TaALMT1-R2, TaALMT1-F2/GFP-R2, SQ-γ1/BS32, and SQ-γ2/BS32 (Supplemental Table S1). The PCR conditions were as follows: 95°C for 2 min, followed by 30 cycles of 95°C for 10 s, 56°C for 30 s, and 72°C for 30 to 60 s. After amplification, amplicon specificity was verified by loading RT-PCR products onto 1% or 2% Tris-acetate-EDTA agarose gels stained with ethidium bromide. Agarose gels were visualized using a UV transilluminator.

β-Galactosidase Enzyme Assay

Leaf tissues (0.25 g) were homogenized under ice-cold conditions in extraction buffer containing 100 mm sodium acetate, pH 4.5, 1 mm EDTA, 0.1 mm DTT, 100 mm NaCl, and 1% (w/v) insoluble polyvinylpolypyrrolidone. The homogenate was centrifuged at 15,000g for 10 min at 4°C. The resulting supernatants were used for enzyme assays. The β-galactosidase activity was determined by measuring o-nitrophenol hydrolysis of the substrate o-nitrophenol-β-d-galactopyranoside. The reaction mixture consisted of 50 mm sodium acetate (pH 4.5) and 5 mmo-nitrophenol-β-d-galactopyranoside in a final volume of 1 mL and was preincubated at 50°C for 1 min. The enzyme (50 µL) was added, and the mixture was incubated with the homogenate solution at 50°C for 5 min and then stopped with 500 µL of 1 m sodium carbonate. The liberated o-nitrophenol from the assay reaction was measured by the A410.

Immunoblot Assay

Total proteins were extracted from leaves or roots of wheat with extraction buffer (100 mm Tris-HCl [pH 7], 2 mm DTT, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1% Triton X-100, 5% [w/v] polyvinylpolypyrrolidone, and 10% glycerol). Plant debris were pelleted by centrifugation at 15,000g for 10 min at 4°C. The total soluble protein extracts from the supernatant (25 µg of proteins) were separated by SDS-PAGE using 12% acrylamide gels. Proteins were transferred onto a polyvinylidene difluoride membrane (Bio-Rad) by electroblotting. The membrane was then incubated in a plastic container with a blocking reagent (5% [w/v] skimmed milk) in TBS with Tween 20 (TBS-T; 25 mm Tris, 0.15 m NaCl, and 0.05% Tween 20, pH 7.5) for 60 min at room temperature. After incubation with a rabbit polyclonal anti-GFP antibody (Abcam; 1:10,000) at 4°C for 16 h, the membrane was washed three times with TBS-T for 10 min and incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG H&L antibody (Cedarlane; 1:10,000) at room temperature for 1.5 h. The membrane was then washed three times with TBS-T for 10 min. The proteins were detected using the Western Lightning Plus-enhanced chemiluminescent substrates (PerkinElmer) at 10 s of exposure with a CCD camera of the FluorChem SP digital imaging system (Alpha Innotech).

Statistical Analysis

Statistical analysis was performed using Instat 3.0 (Graphpad). Values are expressed as means ± sd. Means were compared using one-way ANOVA followed by Tukey’s posthoc test. Significance was set at P ≤ 0.05.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under the following accession numbers: TaALMT1.1 (AB081803) and BSMV ND18 RNA γ (U13917.1).

Acknowledgments

We thank Dawei Li (State Key Laboratory of Agrobiotechnology, China Agriculture University) and Shawn Clark (National Research Council Canada) for providing the BSMV pCaBS-α, pCaBS-β, and pCaBS-γ, A.L.N. Rao (University of California, Riverside) for providing the pCass4-Rz vector, and F. Ouellet for critical reading of the article and for sharing the CFX96 Touch Thermal cycler (Bio-Rad).

Footnotes

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Mario Houde (houde.mario{at}uqam.ca).

A.C. performed the experiments; A.C. and M.H. designed the experiments, interpreted the data, and wrote the article.